diversity in chemotaxis mechanisms among the bacteria and ...bacteria and archaea 23, 33, 64, 67,...

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, June 2004, p. 301–319 Vol. 68, No. 21092-2172/04/$08.00�0 DOI: 10.1128/MMBR.68.2.301–319.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Diversity in Chemotaxis Mechanisms among the Bacteriaand Archaea

Hendrik Szurmant and George W. Ordal*Department of Biochemistry, Colleges of Medicine and Liberal Arts and Sciences,

University of Illinois, Urbana, Illinois 61801

INTRODUCTION .......................................................................................................................................................301SIGNAL RECOGNITION AND TRANSDUCTION ..............................................................................................302

Receptors..................................................................................................................................................................302Classes of receptors............................................................................................................................................302HAMP domain.....................................................................................................................................................304Methylation of class I and III receptors .........................................................................................................304Structure of receptors ........................................................................................................................................305Effect of attractant on structure .......................................................................................................................305Maturation by CheD ..........................................................................................................................................306Binding proteins..................................................................................................................................................306Oxygen sensing....................................................................................................................................................306

Phosphotransferase System...................................................................................................................................306EXCITATION..............................................................................................................................................................306

CheA Kinase ............................................................................................................................................................306Coupling to receptors .........................................................................................................................................307

CheY Response Regulator .....................................................................................................................................308Signal Amplification ...............................................................................................................................................309

ADAPTATION.............................................................................................................................................................309Methylation..............................................................................................................................................................309

CheR methyltransferase.....................................................................................................................................309CheB methylesterase ..........................................................................................................................................310

Methylation-Independent Adaptation ..................................................................................................................311CheV adaptational and coupling protein ........................................................................................................311CheC dephosphorylating and adaptational protein.......................................................................................311

SIGNAL REMOVAL ..................................................................................................................................................311CheZ Phosphatase ..................................................................................................................................................313CheC/FliY/CheX Phosphatase...............................................................................................................................313Phosphate Sink .......................................................................................................................................................313

MULTIPLE COPIES OF CHEMOTAXIS GENES ...............................................................................................313LOCALIZATION OF CHEMOTAXIS PROTEINS ...............................................................................................314CONCLUSIONS AND PROSPECTS.......................................................................................................................314ACKNOWLEDGMENTS ...........................................................................................................................................314REFERENCES ............................................................................................................................................................314

INTRODUCTION

The discovery of microbes by Antonie van Leeuwenhoeik(59) was aided by their ability to swim, clearly indicating thatthey are living organisms. Not surprisingly the mechanism con-trolling this behavior has since then been studied extensively.Arguably, chemotaxis is the best understood of all signal trans-duction systems that control movement. While the motilityapparatus differs among organisms, the general control mech-anism is conserved throughout all bacteria and archaea. Thecenterpiece of this control mechanism is the “two-component”system in which phosphorylation of a response regulator re-flects phosphorylation of a histidine autokinase that sensesenvironmental parameters (117). This is the most common

type of signal transduction system in bacteria and controlsdiverse processes such as gene expression, sporulation, andchemotaxis. In chemotaxis, events at the receptors control au-tophosphorylation of the CheA histidine kinase, and the phos-phohistidine is the substrate for the response regulator CheY,which catalyzes the transfer of the phosphoryl group to a con-served aspartate (for a recent review, see reference 250). Theresulting CheY-P can interact with the switch mechanism inthe motor (42, 149, 186, 193, 234). This interaction causes achange in behavior, such as in direction or speed of rotation offlagella. Thus, for example, in Bacillus subtilis, binding of theattractant asparagine to the receptor McpB quickly increasesthe levels of CheA-P and CheY-P, as the excitation event, andproduces increased counterclockwise (CCW) rotation of theflagella (265). The receptors undergo adaptation, a featurethat allows the mechanism to reset so that bacteria canprogress up concentration gradients of attractants or downconcentration gradients of repellents (152). In general, the

* Corresponding author. Mailing address: Department of Biochem-istry, 190 Med. Sci. Building, University of Illinois, Urbana, IL 61801.Phone: (217) 333-9098. Fax: (217) 333-8868. E-mail: [email protected].

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excitation process is highly conserved and there is considerablevariety in the adaptation process (Fig. 1).

Besides these two core proteins, many other proteins con-tribute to making the process work. Chemotaxis proteins canbe ordered into four groups—a signal recognition and trans-duction group, an excitation group, an adaptation group, and asignal removal group (to dephosphorylate CheY-P). The signalrecognition and transduction group includes the receptors (9,81, 118) and ligand binding proteins (4, 86), which are capableof binding effectors outside the cell; a few receptors, however,are cytoplasmic (92, 93, 229). The signal, i.e., changing con-centrations of a chemical, is then transduced to the excitationproteins, CheA and CheY (34, 90). Adaptational proteins alterCheA activity to reset the system. This can be done either byinfluencing CheA activity directly or through the receptors.Lastly, the signal removal proteins ensure that CheY-P levelscan be adjusted to prestimulus levels quickly (the roles of thechemtaxis proteins are summarized in Table 1).

All biochemical processes described here were first discov-ered in the enteric bacteria Escherichia coli and Salmonellaenterica serovar Typhimurium. Fortunately, the enteric chemo-taxis system turned out to be comparatively simple. Since then,chemotaxis in many diverse organisms has been studied andmany, often complex, variations have been found. This reviewaims to summarize and compare the different chemotactic sys-

tems that have been studied to date. The main focus is not E.coli chemotaxis, since many review articles have dealt with itschemotaxis, but Bacillus subtilis, which is arguably the secondbest understood chemotactic bacterium. Other than cheZ, itpossesses at least one copy of each chemotaxis protein found todate (although some fusions of chemotaxis proteins exist thatare not found in B. subtilis).

As will become apparent, the chemotaxis mechanism in B.subtilis is probably close to that of the ancestral organism fromwhich the bacteria and archaea descended, so that understand-ing this mechanism should provide considerable insights intomechanisms used in the diverse species of motile bacteria andarchaea alive today. To appreciate the divergence in chemo-taxis, we tried to include in this review some information aboutat least one representative of each phylum of bacteria andarchaea in which a CheA homolog could be found, indicatingthe existence of a chemotaxis pathway. Of course, many or-ganisms have not yet been studied in detail, and the availableinformation is often based only on the genomic sequence ofthose organisms. We have made a special effort to includeinformation about organisms whose chemotaxis mechanismappears to diverge from the E. coli paradigm. Other reviews,most of which emphasize the E. coli mechanism, include ref-erences 13, 39, 61, 224, and 227. The review by Berg (20) doesjustice to the rather considerable literature dating from the late19th and early 20th centuries.

SIGNAL RECOGNITION AND TRANSDUCTION

Receptors

Understanding how receptors control the CheA kinase is atthe heart of understanding chemotaxis. In E. coli, binding ofattractant inhibits the CheA kinase (34), whereas in B. subtilis,binding of attractant stimulates the CheA kinase (64, 67). It isimportant to understand the structure of receptors and howbinding of attractant (or repellent) changes the activity of theassociated CheA kinase.

Classes of receptors. The receptors are usually transmem-brane proteins with an extramembrane sensing domain thatbinds attractant across the dimeric interface (253), two trans-membrane (TM) regions (TM1, between the N terminus andthe sensory region, and TM2, between the sensory region andthe cytoplasmic regions), and several cytoplasmic regions.These include the signaling region, where the CheA kinase andthe CheW coupling protein and analogs bind, and the meth-ylation region, where methylation/demethylation of the recep-tors occurs to compensate for changes in CheA kinase activitycaused by binding attractant (Fig. 2). The enzymes catalyzingthese reactions, the CheR methyltransferase and the CheBmethylesterase, are described below. Between the methylationregion and the membrane is the HAMP (histidine kinase,adenylyl cyclase, methyl-accepting chemotaxis protein, andphosphatase) linker, which conveys the signal of attractantbinding to the rest of the cytoplasmic region (see below).Based on crystal structures of the extramembrane N-terminalpart of Tar, the aspartate receptor of E. coli, and of the cyto-plasmic C-terminal part of Tsr, the serine receptor, virtuallythe entire receptor is thought to consist mainly of �-helix (51,108, 109).

FIG. 1. General chemotaxis model. A schematic of the biochemicalprocesses in the two-component chemotaxis pathway is shown. Hexa-gons represent response regulator domains. The universal componentsare in red; almost universal components are in orange; optional com-ponents are in yellow.

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Le Moual and Koshland (124) showed that the chemotaxisreceptors in the bacteria and archaea fell into three classes,based on the presence or absence of two pairs of insertions(called “indels,” for insertions/deletions) of 14 amino acids(four turns of the �-helix). The class III receptors are likely tobe the ancestral receptors (124). The locations of these pairs inB. subtilis McpB are illustrated in Fig. 2. They lie on themembrane-proximal side of the signaling regions and methyl-ation regions. The class III receptors have both pairs, the classII receptors have only the pair between the signaling andmethylation regions, and the class I receptors have neither.More recent analysis of sequences in the database (Fig. 3) givessome additional perspective. The original receptors are obvi-ously the class III receptors, but they have undergone modifi-cations early in the lines of descent. One modification, whichpresumably occurred after the gram-positive bacterial line had

diverged from the original line and before the proteobacterialline had diverged, was the deletion of the first and fourth indelsto produce the class II receptors. The class I receptors,which involve deletion of indels 2 and 3, may have arisenseveral times, once during formation of the �-proteobacte-rial line (see Rhodobacter sphaeroides), once during forma-tion of the �-proteobacterial line (see Myxococcus xanthus),once during formation of the �-proteobacterial line (seeRalstonia solanacearum), and once after the �-line had beenformed (see E. coli and Pseudomonas aeroginosa) (Fig. 3).Alternatively, and more probably, the class I receptors mayhave arisen fewer times and, early during the evolution of aparticular line of descent, may have entered by gene transferand displaced the original receptors. One obvious case ofgene transfer is in Clostridium acetobutylicum, a gram-posi-tive bacterium having 38 class III receptors (all the rest of

TABLE 1. List and description of proteins involved in chemotaxis

Protein by category Activity or role Comment Where found References

Signal recognition andtransduction

Methyl-accepting chemotaxisproteins (MCPs)

Receptors Binds chemoeffectors and transducessignal to CheA. Is methylated anddemethylated on glutamateresidues.

Universal among all chemotacticbacteria and archaea

9, 33, 46, 56, 75, 104, 109,110, 118, 119, 128, 148,171, 179, 197, 199, 208,213, 215, 229, 237, 241,243, 245, 247, 264, 265

CheD Glutaminedeamidase

Role in receptor maturation bydeamidation of particularglutamine residues

All chemotactic archaea, gram-positive bacteria, Thermatoga,and some proteobacteria

67, 122, 182

Ligand binding proteins Ligand recognition Binds chemoeffectors and transducessignal to the receptors.

E. coli, not yet known for otherorganisms

1, 3, 4, 24, 28, 36, 40, 53,65, 85, 86, 88, 118, 141,157, 158, 165, 166, 256,266

ExcitationCheA Histidine kinase Autophosphorylates on histidine

residue; substrate for CheY andother response regulators.


23, 33, 64, 67, 70, 90, 137,151, 156, 185, 218, 233,264

CheW Coupling protein Couples CheA to the receptors. Universal among all chemotacticbacteria and archaea

37, 70, 78–80, 131, 144,187

CheY Responseregulator

Primary response regulator. Interactswith the motility apparatus toinduce change of swimmingbehavior when phosphorylated.


16, 25, 26, 35, 49, 209,211, 218, 244, 259

AdaptationCheR Methyl transferase Methylates glutamate residues on

MCPs; role in adaptation.Almost universal amongchemotactic bacteria andarchaea (exception, H. pylori)

47, 48, 116, 182, 214, 264,265

CheB Methyl esterase Hydrolyzes methyl glutamateresidues on MCPs; role inadaptation. Usually has responseregulator domain.

Almost universal amongchemotactic bacteria andarchaea (exception, H. pylori)

10, 45, 57, 73, 74, 90, 102,103, 106, 108, 113, 115,130, 134, 145, 160, 217,219, 220, 226, 264

CheV Coupling andadaptationalprotein

Couples CheA to the receptors;response regulator domain can bephosphorylated; role inadaptation.

Many bacteria including the E.coli close relative S. entericaserovar Typhimurium; howevernot in the archaea or E. coli

63, 99, 181

Signal removalCheC Phosphatase and

adaptationalprotein

Hydrolyzes CheY-P; also probablerole in adaptation.

All chemotactic archaea, gram-positive bacteria, Thermatoga,and some proteobacteria

112, 182, 183, 190

CheX Probablephosphatase

Homologous to CheC, probably hasthe same function.

The spirochetes, some archaea,some gram-positive bacteria,Thermatoga, and someproteobacteria

69, 77, 137

CheZ Phosphatase Hydrolyzes CheY-P. The �- and �-proteobacteria 29–32, 43, 49, 90, 146,188, 189, 195, 223, 257,261

FliY Phosphatase at theflagellar switch

N terminus homologous to CheC;hydrolyzes CheY-P; integral partof the flagellar switch.

The gram-positive bacteria,some spirochetes, andThermatoga

26, 95, 112, 234

CheY* Phosphate sink Alternative CheY that lowersprimary CheY-P levels by actingas a phosphate sink.

The �-proteobacteria, possiblyothers

172, 191, 196, 212

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the gram-positive bacterial receptors are class III) and oneclass I receptor (Fig. 3). The receptors in the spirocheteBorrelia burgdorferi, here termed class O, are a special case:these receptors are missing indels 1 and 4 but are missingpairs of 21 amino acids where the loss of indels 2 and 3would produce a loss of only a pair of 14 amino acids.

HAMP domain. As implied above, the HAMP domain orlinker is found in a number of different types of proteins.Although there is little sequence identity among HAMP do-mains, they generally have two segments of hydrophobic ami-noacyl residues in a heptameric arrangement characteristic ofamphipathic �-helices joined by unstructured amino acids (11,12). The �-helices are probably in a coiled coil (206). Thepurpose of HAMP domains is generally to convey signals frominput domains to output modules. Mutations in the E. colireceptor Tsr HAMP domain caused locked signal output (thatis, persistently clockwise [CW] or CCW or in between, butswitching rarely) (8).

Methylation of class I and III receptors. Considerable workhas been done on McpB from B. subtilis, which might beconsidered a prototype for the class III receptors. The firstdifference noticed between the class I and III receptors wasthat methanol was released in response to all stimuli in B.subtilis (111, 240) and Halobacterium salinarum (163, 216)whereas it was released from E. coli, the prototype organismfor class I receptors, only after the application of negative

stimuli; methanol evolution in this species was suppressed be-low background levels after the application of positive stimuli(105, 241, 242). Interestingly, methanol is released from theclass I receptors of R. spheroides on addition of attractant(which, as in E. coli, inhibits CheA [196]) (145). There is noconsequence on methanol formation of removing the attract-ant. However, as described below, this organism has multiplecopies of chemotaxis genes. The principal methyltransferaseappears to be CheR2; deletion of cheR1 causes methanol to beproduced after both addition and removal of attractant (145).The related organism, Rhodospirillum centenum, however, be-haved as might have been anticipated from the E. coli prece-dent: a reduction of light intensity, which would cause inhibi-tion of the CheA, caused methanol formation, and increase oflight intensity did not (97).

What is the reason for this difference in methanol forma-tion? In E. coli, methylation of receptors increases CheA ki-nase activity, an adaptational mechanism to compensate forthe decreased activity caused by attractant, and it does notappear to matter which sites become methylated (106, 197,238). By contrast, each of the sites in McpB, the one class IIIreceptor that has been characterized in some detail, appear tohave a different function. Glu630 is demethylated both afteraddition and removal of attractant (265) (provided that thissite is in the methylated form). When it is changed to Asp630,which cannot be methylated (198), the resulting mutant has a

FIG. 2. Schematic of the three classes of chemotaxis receptors. Shown is a representative dimer for each class of chemotaxis receptors.Insertion/deletion regions (indels) are shaded in dark gray. Stars indicate sites of methylation for (from left to right) E. coli Tar, M. xanthus FrzCD,and B. subtilis McpB.


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low prestimulus bias; when attractant is added, it adapts only tothe higher wild-type bias, and when attractant is removed, ithardly adapts at all (265). Glu637 seems crucial for causingadaptation to attractants, and only on removal of attractantsdoes methanol evolve from that site (provided that this site isin the methylated form). Glu371 (encoded as Gln371 but de-amidated (probably by CheD [see below]) does not play a roleduring adaptation to addition or removal of attractant but mayin some way be involved in being a regulator of methylation,since an mcpB Q371A mutant shows poor taxis to high con-centrations of asparagine, similar to a cheB mutant (see below)(M. A. Zimmer and G. W. Ordal, unpublished data). Methanolarises from Glu371 only on addition of attractant (providedthat this site is in the methylated form). Thus, in B. subtilis,each of the positions from which methanol arises has a differ-ent function, and these functions, in the case of McpB (asstated above), cause methanol production from Gln(Glu)371and Glu630 on addition of attractant and from Glu630 andGlu637 on removal (provided that these sites are in the meth-ylated form). The extent to which this rule about particularsites having particular functions applies generally is not known;further experiments are required, and one obvious organismwith which to explore this question would be R. spheroides (seeprevious paragraph). In any case, what had seemed until nowthe general rule that class III receptors produce methanol inresponse to all stimuli and class I receptors do so only inresponse to negative stimuli does not appear to be true (the

case of R. spheroides contradicts this rule). No work of this typehas been done for any class II receptors.

Structure of receptors. The E. coli receptors are stabledimers (153) arranged as trimers of dimers (9). The B. subtilisreceptors are also similarly arranged, and, in fact, the trimersof dimers themselves interact near the outer part of the cyto-plasmic membrane (46), as predicted by Kim et al. (110). X-raystructures of the extramembrane ligand binding domain of theE. coli receptor Tar (194) and the cytoplasmic domain of the E.coli receptor Tsr (109) are available, as well as of the ligandbinding domain of the soluble, cytoplasmic B. subtilis receptorHemAT, in the presence and absence of the natural ligand O2.The dimer that binds O2 (the form that stimulates the CheAkinase) is very symmetrical, and the dimer that is free of O2

shows a distinct conformational change in the Tyr70 of one ofthe two subunits of the dimer (254, 255). As described in detailbelow, the CheR methyltransferase and the CheB methyles-terase appear to be especially active on receptors that have justbound or released attractant and before the compensatingmethylation changes have occurred that would help bringabout adaptation. The structural basis of this conformation ofincreased susceptibility is still unknown (45).

Effect of attractant on structure. One longstanding questionhas been how attractant induces the receptors to change CheAkinase activity. In E. coli, attractant causes diminished CheAactivity (35). Based on evidence from work on Cys-substitutedreceptors, whose cross-linking is accelerated by oxidant, there

FIG. 3. Phylogenetic tree of chemotactic bacteria and archaea. The phylogenetic tree was generated from the 16S rRNA sequences by usingthe programs CLUSTALW and DRAWTREE. Included is information regarding the number and class of chemoreceptors for each respectiveorganism. ND, not determined. Organisms, by phylum, are as follows: Archaea: Archaeoglobus fulgidus, Halobacterium salinarum, Methanosarcinamazei, and Pyrococcus abyssi; Thermotogales: Thermotoga maritima; spirochetes: Borrelia burgdorferi, Leptospira interrogans, and Treponemapallidum; cyanobacteria: Nostoc and Synechocystis spp.; gram-positive bacteria: Bacillus subtilis, Clostridium acetobutylicum, Listeria innocua, andThermoanaerobacter tengcongensis; proteobacteria (�-subgroup): Rhodobacter sphaeroides and Sinorizhobium meliloti; proteobacteria (�-subgroup):Ralstonia solanacearum; proteobacteria (�-subgroup): Myxococcus xanthus; proteobacteria (ε-subgroup): Helicobacter pylori; proteobacteria (�-subgroup): Escherichia coli, Pseudomonas aeroginosa, and Vibrio cholerae.


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appears to be “downward piston” movement of TM2 of onemonomer of the receptor dimer (50, 94, 123). Based on exper-iments where the nitroxide spin label was attached to Cysresidues in Cys-substituted receptors, the extent of movementwas deduced to be small, only about 1 A (171). The effect ofrepellent on these receptors and the effect of attractant orrepellent on class III receptors, such as McpB of B. subtilis,where attractant activates rather than inhibits the receptor(67), is unknown.

Maturation by CheD. CheD is a deamidase that deamidatesparticular glutamines in the B. subtilis receptors (122), a func-tion carried out by CheB in E. coli. Most chemotactic bacteriaand archaea carry cheD. Therefore CheD is probably the an-cestral mechanism of glutamine deamidation. The role of thisfunction is not yet fully understood. However without CheDthe receptors are undermethylated and activate the kinasepoorly, indicating that deamidation is necessary for activationof the receptors (67, 182).

Binding proteins. Although in most instances chemoeffec-tors are thought to interact directly with the receptors, in someparticular instances, specifically dedicated binding proteinsbind the chemoeffector and the complex then binds the recep-tor. Thus, in E. coli, galactose binds to the galactose bindingprotein (86), ribose binds to the ribose binding protein (4), andthe complex binds to the receptor Trg (83, 87). Maltose bindsto the maltose binding protein (85), and the complex binds tothe aspartate chemoreceptor Tar (176). It is suspected that theB. subtilis receptor McpC binds attractants indirectly, via bind-ing proteins, since it mediates taxis to all amino acids exceptasparagine, some (such as proline and alanine) at very lowconcentrations (159, 168); however, no mutants in any suchputative binding proteins have been identified.

Oxygen sensing. The oxygen sensor in B. subtilis is HemAT,which is homologous to myoglobin (92, 93). It is similar to therepellent oxygen sensor, also termed HemAT, in the archeonH. salinarum. It is a soluble receptor, having no transmem-brane region, and hence senses the internal oxygen concentra-tion. H. salinarum has another receptor for oxygen as an at-tractant. However, this receptor is homologous to cytochromeoxidase of mitochondria (44) and has six membrane-spanningregions and may be a heme protein that also senses oxygendirectly. Conversely, Aer, the oxygen sensor of E. coli, bindsflavin adenine dinucleotide (FAD) (21, 22, 177), and the signalcaused by changing oxygen concentrations is probably medi-ated by changes in the level of reduction/oxidation of this FAD(21, 177, 235, 236), a process involving a PAS domain in thereceptor (178). Pseudomonas putida would appear to use thesame mechanism (161). Tsr in E. coli also mediates oxygentaxis, perhaps by sensing changes in the proton motive forceacross the cytoplasmic membrane (177, 204) but certainly notby binding oxygen directly (Fig. 4).

Many other organisms perform aerotaxis (reviewed in ref-erence 236). Azospirillum brasilense, an �-proteobacterium, ac-cumulates in an oxygen gradient at 3 to 5 �M. At both lowerand high oxygen tensions, the proton motive force is lower, sothat it is assumed that both positive and negative aerotaxis,which causes accmulation of bacteria at the optimum oxygentension, is due to sensing of changes in the proton motive force(262). R. sphaeroides also accumulates at an optimum oxygenconcentration. It shows negative aerotaxis due to interaction

between the chemotaxis machinery and the Prr system, whichmonitors electron flow through the alternative high-affinitycytochrome oxidase, cbb3, and positive aerotaxis by interactionwith another, unknown sensor, both of which may operatethrough one of the chemotaxis kinases of the cell, CheA2 (180).Some species, for example Sinorhizobium meliloti, do not seemto respond to oxygen in the same way that E. coli and B. subtilisdo (by modulating the frequency of CCW versus CW rotation)but, rather, change their swimming speed in response to oxy-gen gradients (263).

Phosphotransferase System

The phosphotransferase system (PTS) helps mediate taxis toa number of sugars and sugar alcohols in B. subtilis (68, 121,169). Transport is required, but metabolism is not. Unlike taxisto PTS sugars in E. coli, which does not require a specificreceptor and works even when the methylation system is inac-tivated by mutation (162) (but works poorly unless some re-ceptor is present [136]), chemotaxis to PTS substrates in B.subtilis requires the C-terminal part of McpC (Fig. 5). In theseexperiments, chimeras between the asparagine receptor McpBand the proline receptor McpC revealed that the N-terminal,extramembrane part of the receptors mediated amino acidtaxis, as expected, but only the C-terminal part of McpC couldmediate taxis to PTS substrates and, in particular, the methyl-ation region appeared to be involved. The data were bestinterpreted by a model in which unphosphorylated enzyme Iinteracted with McpC to bring about increased CheA activityand adaptation occurred through the normal means (121).Indeed, methanol was produced on addition of glucose, a signthat CheB was stimulated to help bring about adaptation (239).In E. coli, since unphosphorylated enzyme I interacts withCheA (135), it is suspected that interaction of CheA withunphosphorylated enzyme I inhibits CheA. Large changes inthe levels of unphosphorylated enzyme I compared with phos-phorylated enzyme I occur during chemotactic excitation(136). It would seem likely that the requirement for receptorfound by Lux et al. (136) might be due to the inherent lowactivity of CheA in the absence of receptors (34) rather thanthe interaction with a specific receptor, as found for B. subtilis.

EXCITATION

With the exeption of Mycoplasma gliding motility, it appearsthat bacterial and archaeal motility is universely controlled bythe two-component system of the CheA kinase and the CheYresponse regulator.

CheA Kinase

The central enzyme that mediates input, usually as sensed bythe receptors, and creates an appropriate signal for the motoris the CheA kinase. Attractants inhibit it in E. coli (34), S.meliloti (192), and R. spheroides (196) and stimulate it in B.subtilis (67). As described in “CheY response regulator” (be-low), it is likely that for the archaea and the spirochetes, pos-itive stimuli (for instance, chemoattractants or attractant light),decrease CheA activity. Thus, B. subtilis would appear to be the


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exception to this rather incompletely verified hypothesis thatpositive stimuli decrease CheA activity.

On activation, CheA becomes phosphorylated on a particu-lar histidine residue (His46 in B. subtilis and His48 in E. coli[258]). CheA has five domains (described in detail in reference23), and this His residue is located within the first, P1 or Hpt,domain (156). The fourth (P4) domain is where ATP binds andcatalysis occurs. The third (P3) domain is the dimerizationdomain, important since CheA is a dimer that transphospho-rylates (232) (the P4 region of one monomer phosphorylates aHis residue in the P1 domain of the other monomer). The P5domain is where CheA contacts the receptors and the couplingprotein, CheW. The P2 domain is where CheY and CheB,which receive phosphoryl groups from CheA-P, dock (23, 89,233). The exact mechanism of CheA autophosphorylation isnot yet known; however, several conserved regions within theP4 domain—the N-box, G1-box, F-box, G2 box, and GT-block—are essential for catalysis in E. coli and are thought tobe involved in positioning of ATP into the active site (Fig. 6)(91).

CheA-P from B. subtilis differs from its E. coli counterpart in

being of considerably lower energy (Keq � 1.2 � 104 instead of1 in the reaction CheA � ATP3 CheA-P � ADP) (66). WhenE. coli becomes somewhat deenergized, it becomes smoothswimming (107), since CheA cannot be phosphorylated, andthus the bacterium has a larger “diffusion constant” so that itwill leave the local environment by rapid translational move-ment. However, when B. subtilis becomes somewhat deener-gized, the chemotaxis system still functions. Thus, the bacteriado not become tumbly (the condition in the absence of CheY-P[27]) and thus unable to move away from the unfavorableenvironment but instead use chemotaxis to depart, a muchmore effective process than unregulated smooth swimming. Interms of phylogeny, CheA from B. subtilis clusters with CheAsfrom archaea and spirochetes, apart from CheAs of the pro-teobacteria (2).

Coupling to receptors. CheA is coupled to the receptors viaCheW. CheW is present in all bacteria that have chemotaxis orphototaxis receptors. A second protein, CheV, which is aCheW-CheY fusion protein, is also capable of coupling thekinase to the receptors and is described below. CheV is present

FIG. 4. Aerotaxis receptors. Shown is a schematic of the four known types of aerotaxis receptors. Indirect aerotaxis defines receptors that detectoxygen levels by the proton motive force or redox state of the cell. Direct aerotaxis defines receptors that detect levels directly by interacting withoxygen. Black diamonds represent the indicated receptor cofactors.


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in many eubacteria, including S. enterica serovar Typhimurium,a close relative of E. coli, but not in E. coli and not in anyarchaea. In E. coli, CheW is required for activation of CheAbut not for its inhibition (7, 35). CheW is homologous to the P5or regulator domain of CheA (23).

CheY Response Regulator

The primary response regulator that governs the direction offlagellar rotation is CheY-P (16, 25, 90) and, as implied above,it causes CCW flagellar rotation in B. subtilis and CW flagellarrotation in E. coli. It catalizes its own phosphorylation on a

conserved aspartate residue by using CheA-P as a substrate.This process is thought to involve several conserved residues—two aspartates, which position an essential Mg2� ion, a lysine,and a threonine (Fig. 6) (132, 244, 260). In flagellated bacteria,CheY-P interacts with FliM, shown for E. coli (248) and B.subtilis (26, 234). In the spirochete Treponema denticola, mu-tation of cheA blocked chemotaxis and caused the bacteria tohave few reversals of motion (137), implying that CheY-Pcauses reversals of motion. To achieve this, it probably binds toFliM, which is present in the spirochetes. The archeon H.salinarum similarly showed no reversals of motion when cheAor cheY was deleted and, indeed, showed preferential forward

FIG. 5. PTS in chemotaxis. The two known chemotaxis pathways for PTS sugars are shown. Transport of PTS sugars increases the concentrationof unphosphorylated enzyme I (EI) that can either directly interact and inhibit CheA (receptor-independent system), as is the case in E. coli, orindirectly stimulate CheA through the receptors (receptor-dependent system), as thought to be the case in B. subtilis. The letters A, B, and Crepresent components of an ABC transporter. For better understanding, PTS proteins are in yellow, B. subtilis chemotaxis proteins are in red, andE. coli chemotaxis proteins are in green.


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swimming (chronic CW rotation of the flagella at each end ofthe archeon) (184). Thus, in this organism, CheY-P also causesreversals of motion and is required for CCW rotation of theflagella. (Note: these are right-handed flagella [5], not themore common left-handed flagella [139] found in E. coli and B.subtilis, and the flagella themselves are more similar to type IVpili [52] than to the flagella of bacteria.) There are no FliMhomologs in the archaea, and the site of the interaction ofCheY-P to control the direction of flagellar rotation is un-known. The implication of these findings is that the flagella ofHalobacterium and spirochetes have a default direction of ro-tation in the absence of CheY-P, as do the flagella in theperitrichous bacteria like E. coli and B. subtilis, and thatCheY-P not only facilitates rotation in the opposite directionbut also facilitates switching between the two directions. Howthis might occur is mentioned below (see “CheC dephospho-rylating and adaptational protein”).

Signal Amplification

Binding of one or two receptors by attractant can lead to abehavioral response in B. subtilis (113) and E. coli (195). Usingphotoreleased aspartate, Jasuja et al. (96) found that nanomo-lar asparate (1.2 �M KD) could evoke a response and that theresponse times were proportional to changes in receptor oc-cupany near the threshold, irrespective of prior occupancy.(Therefore, adaptation is complete.) In experiments with E.coli, using fluorescence resonance energy transfer to measureCheY-P levels (rather than CCW/CW rotation, which is acomplex function of CheY-P levels), Sourjik and Berg (210)found that the cheB mutant was very insensitive to attractantcompared to both the wild type and a cheR mutant. Usingphotoreleased asparate, Kim et al. (108) also found that thecheB mutant was far less sensitive than was the wild type. Bothgroups found that absence of CheZ, which catalyzes the de-phosphorylation of CheY-P, had little effect on amplification;therefore, accelerated loss of CheY-P is not the cause of signalamplification; it must be sought in signal generation. HowCheB might be involved in this is described in “CheB methyl-

esterase” (below). Besides this, it seems likely that organiza-tion of the receptors into a lattice could lead to amplification;in this arrangement, judicious methylation of receptors to in-crease CheA activity (after reduction of activity from attractant[in E. coli]) would allow this amplification to occur over abroad range of attractant concentrations (201, 202). However,it would appear that this alleged lattice quickly forms anddisappears, according to circumstances (see “Localization ofchemotaxis proteins,” below).

ADAPTATION

To sense ever higher concentrations of attractant and tomove toward favorable environments, chemotaxis systems haveto be able to adapt to existing stimuli. Additionally, the natureof bacterial motion requires the ability to recognize when thebacterium is moving in the wrong direction, i.e., away fromhigher attractant concentrations. To do that, a “memory” isrequired that is able to indicate whether higher or lower con-centrations are being reached (120). This is achieved by theadaptational mechanisms. The methylation system of CheRand CheB is the only adaptational mechanism in E. coli thathas been studied, although another, undescribed mechanismmay exist (162, 228). However, other organisms, e.g., B. subtilis,have at least partly characterized adaptational systems,namely, CheV and CheC (Fig. 7), in addition to the mechanisminvolving CheR and CheB (99, 112).

Methylation

CheR methyltransferase. CheR methyltransferase transfersmethyl groups from S-adenosylmethionine to particular gluta-mate residues (102, 164, 238) on the receptors (82, 164), withproduction of S-adenosylhomocysteine (47, 48, 214). In B. sub-tilis, it is required for adaptation to repellents. In its absence, B.subtilis is very tumbly (with predominantly CW rotation of theflagella) (116). In E. coli, it is required for adaptation to at-tractants (76, 119) and binds both to a flexible tether at theC-terminal end of the receptor and to the methylation region

FIG. 6. Schematic of CheA and CheY. Shown are the histidine kinase CheA and the general response regulator CheY. For CheA, the fivedomains are labeled P1 through P5. The phosphoreceiving histidine in P1 is highlighted in gray, and conserved regions within domain P4 that arethought to play an active role in catalysis are also highlighted in gray. For CheY, conserved residues that participate in catalysis are positioned asindicated.


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of the same or a nearby receptor and so moves through areceptor cluster in a hand-over-hand fashion (58, 127, 252).This property, as might be expected, allows CheR bound toone receptor to methylate another (125, 126, 129), and recep-tors lacking the C-terminal binding site are poorly methylatedunless receptors containing it are present in the same cell (19).The properties of E. coli CheR and B. subtilis CheR are likelyto be similar since receptors of either organism can be meth-ylated by CheR from either organism (47). CheR is present invirtually all bacteria and archaea showing chemotaxis, since itis important for adaptation; the exception is Helicobacter pylori,which has CheV, whose phosphorlation, as described below, isknown to help bring about adaptation in B. subtilis (99).

CheB methylesterase. Unlike CheR, CheB is usually regu-lated; it has an N-terminal response regulator domain, subjectto phosphorylation, and a C-terminal enzymatic domain (57,90, 134, 221). The E. coli enzyme is 10-fold more active whenphosphorylated (134). The B. subtilis enzyme can satisfy therequirement for methylesterase for chemotaxis in E. coli (115),and, in vitro, both enzymes were able to demethylate both B.subtilis and E. coli receptors (160). One interesting possibledifference is that CheB deamidates particular E. coli receptors,after which the site can be methylated and demethylated (102).In the instances where B. subtilis receptors are deamidated (forinstances where the reaction has been characterized), this re-action is catalyzed by CheD, not by CheB (122). Perhaps, then,it is not surprising that the B. subtilis CheB appears to be moreclosely related to CheB in the archaea, which have CheD andclass III receptors, than to CheB in most bacteria, some ofwhich lack CheD and, in the case of the proteobacteria, haveclass I and class II receptors (2).

The isolated enzymatic domain of CheB catalyzes receptordemethylation in B. subtilis (45) and in E. coli (134), althoughnot as effectively as does the phosphorylated whole enzyme(10, 57). However, in B. subtilis, a truncated cheB encoding the

enzymatic domain complements a null cheB mutant, and thisstrain (null cheB mutant having truncated cheB on a plasmid)releases enhanced levels of methanol on both addition andremoval of attractant. This result implies that the demethyl-ation reaction involves primarily the existence of a suitablesubstrate. The value of phosphorylation of CheB would thenbe to increase the rate of receptor demethylation and thusspeed up adaptation and to minimize unnecessary receptordemethylation, since loss of a methyl group is equivalent tohydrolyzing 11 to 14 ATP molecules to ADP and Pi (45, 225).Interestingly, CheB from Campylobacter jejuni lacks a responseregulator domain (142). It is assumed that the time duringwhich enhanced methanol formation occurs is the time be-tween addition or removal of attractant and the resulting com-pensating (to bring about adaptation) methylation events onthe receptor. This particular susceptible conformation does notrequire the coupling proteins CheW or CheV for events afterthe addition of attractant but does require them for eventsafter the removal of attractant (45). The fact that the receptorsthat have bound attractant are more susceptible to methyles-terase was shown in vitro many years ago (160).

The methylation system, involving CheR and CheB, is im-portant for chemotaxis to high concentrations of attractant andonly peripherally for chemotaxis to low concentrations of at-tractant in B. subtilis (113, 115). The reason is thought to bethat at low concentrations of attractant, signal amplificationoccurs since binding attractant to a receptor activates neigh-boring receptors (203). At high concentrations of attractant,without CheB to generate charge-charge repulsion, there areno available free receptors that can activate the kinase onbinding attractant (113). At low concentrations of attractant,methylation-independent systems suffice to bring about adap-tation, such as the CheV system (see below). Thus, it may notbe surprising that the amount of methanol evolved increasesexponentially with receptor occupancy by attractant (111).

FIG. 7. Adaptation systems. A flowchart of the possible means of adaptation is shown. The almost universal methylation-dependent adaptationsystem is shown on the left. The less highly conserved methylation-independent pathways are on the right. We speculate here that CheV mightdirectly influence CheA activity following phosphorylation, as shown, while the means of CheC activation and adaptational action are not yetunderstood. Black diamonds represent a chemoeffector. Adaptational proteins are hatched; excitatory proteins are shaded.


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CheB may play another role as well. Under certain condi-tions, it appears to be required for a response to removal ofattractants (113), and the first characterized cheB (then termedcheL) mutant (OI1130) was tumbly and unresponsive to stim-uli (167). The implication is that this mutant CheB preventsnormal functioning of the receptor-CheA complex. No similarmutants have been characterized in other organisms, such as E.coli, but absence of CheB results in an E. coli strain that is farless sensitive to the addition of attractant than is the wild type(108, 210), as described in more detail in “Signal amplification”(above). As stated above, attractants inhibit CheA in E. coli;this experiment implies that CheB is needed to amplify theprocess leading to low CheA-P and thus CheY-P levels (18).One way of achieving this is to inactivate the CheA associatedwith a receptor complex and have CheB molecules, whichundergo very rapid autodephosphorylation (90), diffuse toneighboring complexes and inactivate them.

Methylation-Independent Adaptation

CheV adaptational and coupling protein. CheV has twodomains, a N-terminal domain homologous to CheW and aresponse regulator C-terminal domain (63), and can substitutefor CheW in coupling receptors to CheA in B. subtilis (181).Insight into its function has come from experiments using amutant in which the phosphorylated aspartatyl residue wasreplaced with an alanyl residue (CheV D235A strain) and thewhole response regulator domain was deleted. Both mutantstrains showed poor adaptation to the addition of attractants,a result implying that the purpose of CheV phosphorylation isto bring about adaptation (99). Interestingly, mutants lackingcheV altogether did adapt normally in the tethered-cell assay.Thus, it would appear that the conformation of the coupling(“CheW”) domain of CheV is such as to strongly favor recep-tors bound with attractant in the conformation to activateCheA, since other adaptation systems like the methylationsystem are unable to restore the prestimulus bias. Since theCheV D235A strain adapts poorly, it would seem that adapta-tion requires phosphorylation of D235, probably so that theregulator domain can interact with the coupling domain toaffect the conformation of the coupling domain and allow theattractant-bound receptors to reassume their prestimulusconformation.

CheV may be the only adaptation system in H. pylori, sinceCheR and CheB are absent. However, there are three CheVs,of which only CheV1 appeared to be required for chemotaxis,and none could substitute for CheW (172).

CheC dephosphorylating and adaptational protein. As men-tioned below CheC has CheY-P hydrolyzing activity (234a).However it is hard to explain the tethered cell phenotype of aB. subtilis cheC mutant, other than by assuming that it alsoplays a role in adaptation. While the prestimulus rotationalbias of cheC is approximately that of the wild type, cells donot adapt to the addition of attractant (112, 182). This can beexplained by the presence of persistently elevated CheA-Plevels. CheC was shown to bind to McpB and CheA and somight either directly or indirectly influence CheA activity(112).

In addition, mutants with mutations in cheC have a lowerfrequency of switching the direction of rotation (from CCW to

CW and from CW to CCW), implying that CheC lowers theenergy of transition of switching (thus, the wild type, which hasCheC, has a higher switching frequency than does the cheCmutant). Mutants with mutations in cheB have the oppositephenotype, an increased frequency of switching (190). It ishard to imagine that CheB binds to the switch but not sofar-fetched to imagine that CheC does, since it is homologousto most of the FliM and FliY proteins (two of the three pro-teins comprising the switch). The state of two proteins beinghomologous does not necessarily imply that they bind eachother; however, such binding does occur, for instance betweenCheA and CheW, the P5 domain of CheA being homologousto CheW (see above). These results can be accounted for byassuming that CheC has minimal affinity for overmethylatedreceptors so that in a cheB mutant, there would be more CheCbound at the switch. Very interestingly, a cheB mutant of H.salinarum shows increased frequency of reversals, with no ef-fect on the ratio of CW and CCW rotation of the flagella,compared with the wild type (184). Similarly, the cheB mutantof B. subtilis has a normal bias (i.e., the same ratio of CW andCCW rotation of the flagella as in the wild type). Thus, it is nothard to imagine that reversal frequency in H. salinarum and B.subtilis is controlled by the same mechanism, namely, theamount of CheC bound at the switch. Presumably, in H. sali-narum, the mechanism by which CheY-P produced by repel-lents or repellent light would cause increased reversals wouldinvolve inducing increased CheC binding at the switch. A sim-ilar situation may exist for the spirochetes, which undergoreversals of motion, except that there the CheC homolog isCheX, which is smaller than CheC (137).

CheC is not the only substance, however, that affects theswitching frequency. Fumarate also promotes increasedswitching frequency in E. coli (17, 155, 175) and also increasesthe probability of CW rotation (154) by binding at the flagellarswitch (175). As a central metabolite, fumarate would not beexpected to be a chemotaxis signal whose concentrationchanges on a timescale of seconds, as does CheY-P, but might,instead, somehow be a barometer of the metabolic state of thecell. However, the way in which it would facilitate cell survivalby reducing bias and switching frequency when present at lowconcentration and increasing bias and switching frequencywhen present at high concentration is, at this point, unknown.

SIGNAL REMOVAL

One of the unique challenges faced by chemotaxis systems isthe necessity for quick responses (on a timescale of seconds) toever changing environments. This is in constrast to most othertwo-component signal transduction systems that control geneexpression and act over minutes to hours. To cope with thisproblem, the half-lives of CheY-Ps, are brief, shorter than 1min (38, 66). CheY is thought to actively catalyze autodephos-phorylation, a process involving several conserved residues(two aspartates, a lysine, and a threonine) and a Mg2� ion(133, 205, 222). However, the half-lives still appear to be toolong. To further speed the signal removal, the enteric organ-isms as well as some other �- and �-proteobacteria expresscheZ. The protein further destabilizes CheY-P (257). In theseorganisms, CheZ is essential for chemotaxis. It was puzzlingthat most chemotactic bacteria and archaea do not carry a


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cheZ gene. Based on data available for B. subtilis, it is nowapparent that in many if not most of these organisms a com-bination of CheC, FliY, and/or possibly CheX perform thisfunction (234, 234a). These three proteins are homologous buthave no sequence similarity to CheZ (Fig. 8). A third mecha-nism of signal removal has been suggested for S. meliloti and

other �-proteobacteria where an alternative CheY is thoughtto act as a phosphate sink and possibly support signal removal(212). The methods of signal removal for different bacteria aresummarized in Fig. 9. Interestingly, some organisms do notexpress a cheZ or cheC homologue or an alternative cheY.Other means of signal removal in these organisms could in-

FIG. 8. Schematic of CheY-P-hydrolyzing proteins. For CheZ, the C-terminal CheY-P binding region is shown in black and the area includingwhat is thought to be the active site is shaded in gray. For FliY, the CheY-P binding site is shown in black. For FliY, CheC, and CheX, conservedregions are in gray, with highly conserved residues positioned as indicated.

FIG. 9. Means of CheY-P hydrolysis in chemotactic organisms. The tree was generated as described for Fig. 3. Chemotactic organisms thatencode a CheC homolog are highlighted in light gray; dark gray represents organisms that encode a CheZ; black represents organisms that encodean alternative CheY that acts as a phosphate sink; white represents organisms with no known mechanism of CheY-P hydrolysis. V. cholerae encodesboth CheZ and CheC homologs and is therefore indicated by dark and light gray squares.


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clude the presence of a response regulator domain fused toother chemotaxis components acting as phosphate sink.

CheZ Phosphatase

CheZ is found exclusivly in the �- and �-proteobacteria (seethe legend to Fig. 3 for type species). From its limited spreadamong bacteria it can be concluded that it evolved relativelylate, and that the original chemotactic organism had othermeans of signal removal.

As part of the E. coli chemotaxis system, CheZ is by far thebest studied CheY-P phosphatase and the only one for whichan X-ray structure is available (257). CheZ is localized to thereceptor complexes, in the enteric bacteria, via CheA-short (aform of CheA that lacks part of the N-terminal sequence,including the site of phosphorylation [251]) (209). However,the meaning of this localization is not yet clear, since a mutantnot capable of making CheA-short does not have a chemotac-tic phenotype (150). At one time, it was thought that enhance-ment of CheZ activity might be the means by which CheY-Pcould be rapidly hydrolyzed following addition of attractant togenerate the excitatory signal. However, CheZ does not appearto play any excitatory role (108, 210). Studies of fragments ofthe protein identified the C terminus of CheZ to be theCheY-P binding domain (29). However, insight into the mech-anism of CheZ action remained elusive until the X-ray struc-ture of CheZ in complex with activated CheY was solvedrecently. Based on this structure, it has been proposed thatCheZ residue Gln147 is actively involved in increasing the rateof CheY-P hydrolysis by positioning and activating a watermolecule in the active site of CheY-P (257).

CheC/FliY/CheX Phosphatase

Most chemotactic bacteria and archaea do not encode aCheZ homolog and so must cope with the problem of fastsignal removal in some other way. A recent study found thatthe flagellar switch protein FliY in B. subtilis is able to increasethe rate of CheY-P hydrolysis. The C-terminal region of FliYis homologous to E. coli FliN. The N-terminal domain is ho-mologous to two other chemotaxis proteins, CheC and CheX(234). While FliY is exclusive to gram-positive bacteria, somespirochetes, and Thermatoga, CheC and/or CheX can be foundin almost all phyla of chemotactic organisms, including someproteobacteria. Indeed, B. subtilis CheC shares the ability ofFliY to hydrolyze CheY-P (234a). Therefore, conserved resi-dues between CheC and FliY are possibly involved in thechemistry. Six residues—Asp39, Glu43, Asn46, Ser136,Glu140, and Asn143 (following B. subtilis FliY numbering)—are conserved among these proteins, and any could play asimilar role to residue Gln147 in E. coli CheZ. Interestingly,the latter three residues are also conserved among CheX pro-teins, which appear to be truncated versions of CheC. Mostchemotactic bacteria and all chemotactic archaea have a CheChomolog. Whether the mechanism of dephosphorylation ofCheY-P is similar to that of CheZ will ultimately be shown onlyby obtaining an X-ray refraction structure of any of theseproteins in complex with CheY.

Phosphate Sink

Work on the �-proteobacteria S. meliloti and R. spheroidessuggests a third mechanism of signal removal (174, 212). Ineach of these organisms, deletion of at least two CheY ho-mologs causes a defect in chemotaxis. In S. meliloti, CheY2 isthe main response regulator that interacts with the flagellarswitch and causes the reversal of flagellar rotation. CheY1does not interact with the switch, although, like CheY2, it israpidly phosphorylated by CheA-P. CheY2, however, is capa-ble of transferring its phosphoryl group back to CheA andsubsequently to CheY1, so that CheY1 may act as a phosphatesink (212). In R. spheroides this mechanism is more complex,since this organism contains six cheY genes; some CheY pro-teins are thought to act as phosphate sinks (174). Since manychemotactic organisms have more than one cheY, one canimagine that the phosphate sink mechanism may be wide-spread. In addition, some organisms do not encode any of theknown signal-removing proteins and, we speculate, may useresponse regulator domains fused to other chemotaxis pro-teins. However, no data suggesting this have yet been reported.

MULTIPLE COPIES OF CHEMOTAXIS GENES

As mentioned above, the E. coli chemotaxis system is simplein comparison to most other chemotaxis systems. This is be-cause there is only one copy for each chemotaxis protein. B.subtilis already proves more complex since partially redundantproteins like CheW and CheV or like CheC and FliY makephenotypes less severe and conclusions about them less obvi-ous (99, 181, 234). However, still more complex systems can befound. Some organisms contain multiple sets of chemotaxisgenes, some of which may have functions other than control-ling motility. P. aeruginosa has five clusters of chemotaxis genes(62, 230). Two of these clusters (I and V) are required forchemotaxis (100, 146). Another (IV) is required for chemotaxisby twitching motility (55, 101), which involves extension andretraction of type IV pili (207). This type of movement isthought to facilitate movement across surfaces and formationof biofilms (170); it usually involves rafts of cells rather thanindividual cells (147). It seems reasonable that the apparentredundancy of chemotaxis genes in this organism is due togenes within a cluster being devoted to a particular function,such as chemotaxis involving flagella or twitching motility, andis not actual redundancy.

Another organism with multiple copies of chemotaxis-typegenes is M. xanthus. M. xanthus has two types of motility,A-motility and S-motility (98, 200). S-motility is homologous totwitching motility in P. aeruginosa and involves extension andretraction of type IV pili (231). M. xanthus has nine clusters ofchemotaxis-type genes (14), of which the Frz genes mediatechemotaxis by controling reversals of (gliding) cells, the Difgenes are involved in fibril formation (necessary for S-motil-ity), and the Che4 cluster is also involved in S-motility. How-ever, another set, the Che3 cluster, affects the entry of M.xanthus into the developmental program to produce spores,and the output would appear to be the response regulatorprotein CrdA (whose cognate histidine kinase appears to beCheA3), predicted to be the transcriptional activator for 54-dependent promoters (114). Thus, in this case, what must have


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originally been a chemotaxis-type set of genes controlling mo-tility evolved into a set of genes controlling transcriptionalactivation. The main difference between ordinary transcrip-tional activation and this type might be that the latter wouldundergo adaptation so that the time derivative of the inputsignal, rather than the magnitude of the input signal itself,would control transcription. Such an arrangement might pro-vide for sensitivity to changes over many orders of magnitude,as in the case for bacterial chemotaxis (41, 54). A detailedaccount of the issues involved may be found in reference 14.

The �-proteobacterium R. spheroides has three sets of che-motaxis genes. cheOp1 contains cheY1, cheA1, cheW1, cheR1,and cheY2. cheOp2 contains cheY3, cheA2, cheW2, cheW3,cheR2, cheB1, and tlpC. cheOp3 contains cheA4, cheR3, cheB2,cheW4, slp, tlpT, cheY6, and cheA3. Besides these chemotaxisgenes, there is one encoding a fusion protein of CheBRA and13 encoding receptors, including 4 cytoplasmic receptors (lack-ing a membrane-spanning region) and cheY4 (www.jgi.doe.gov/JGI microbial/html/rhodobacter). Deletion of cheA2 preventsaerotaxis, phototaxis, and chemotaxis, but deletion of cheA1

has little effect. Deletion of cheW2 has a much bigger effect onlocalization (see below) of cheA2 at the poles of the cell thandoes deletion of cheW3, which marginally affects cheA2 local-ization. CheA2 causes the phosphorylation of CheY4, andCheY3 facilitates signal temination, possibly acting as a phos-phate sink (196). CheA1, with its cognate response regulatorCheY5, mediates a repellent (“inverted”) response (196). Thefunction of the genes in cheOp3 is unknown. In this organism,the response to negative stimuli is to stop (rather than rotatethe single polar flagellum CW, as does E. coli for its peritri-chous flagella). On stopping, the flagellum goes from helical tocoiled (15). This transition, coupled with rotational Brownianmotion, reorients the bacterium (173) so that the next smoothswim will take a new direction.

LOCALIZATION OF CHEMOTAXIS PROTEINS

Polar localization of chemotaxis proteins was first exploredin Caulobacter, a natural organism with which to investigatepolarity since it undergoes differentiation in which a stalk cellproduces a swarmer cell with a single polar flagellum that ismade shortly before cell division in every generation (249).Later, the flagellum is discarded and is replaced by a new stalk(60). The receptor is located at the pole (6). This expectedfinding led to an unexpected one, namely, that the receptors ofE. coli are also located at the poles of the cell (140). Thisfinding has led to a considerable body of research that hasdocumented that chemotaxis receptors generally are clustered,usually at the pole but, for cytoplasmic receptors as in R.spheroides, at an apparently random place in the cytoplasm (84,143, 246). The B. subtilis asparagine receptor, McpB (and pre-sumably the other receptors spanning the membrane) is alsolocated at the poles of cells (113). However, the significance ofclustering for signal amplification is uncertain, since it wasunaltered in E. coli strains lacking CheR or CheB (138), butstrains lacking CheB are very impaired in sensitivity to attract-ants, although strains lacking CheR are still very sensitive (108,210). Moreover, addition of a multivalent ligand that can bindtwo receptors simultaneously greatly increases the sensitivity ofheterologous receptors to their ligands, and this sensitivity is

diminished when other heterologous receptors are deleted (71,72). Thus, it would appear that clustering of receptors mayfacilitate taxis since receptors are close to each other but activesignaling must require a particular arrangement of the recep-tors, a goal that is hard to achieve when they are fully meth-ylated.

CONCLUSIONS AND PROSPECTS

One great achievement in our understanding of bacterialchemotaxis in the 1970s was the discovery of the methylationsystem as foundational for bringing about adaptation to stim-uli. In the 1980s and early 1990s came the discovery of thetwo-component system involving phosphotransfer as mediatingexcitation. Now, during the past decade, there has been agrowing appreciation of the diversity of chemotactic mecha-nisms used in the broad sweep of bacteria and archaea. In thisreview we have emphasized this diversity. We have acknowl-edged that many of the principles have been worked out in theE. coli-S. enterica chemotaxis system and that great progress inelucidating that system is still occurring. However, it has be-come clear that the E. coli system is streamlined and lacks orhas significantly modified some basic features of the primordialmechanism that existed when the bacteria and archaea sepa-rated during evolution. It seems that many of these featuresexist in the B. subtilis mechanism, and the elucidation of thismechanism has, accordingly, been one of the features of thisreview. The processes used to restore behavioral conditions totheir prestimulus conditions have changed the most during thestreamlining that has led to the E. coli mechanism. However,evolution in other organisms has not stood still, and a lot ofchanges in other directions have occurred since the primordialmechanism was widely used; we have tried to do justice tothese. A lot of information, however, is still at the genome-sequencing level, and more behavioral, genetic, and biochem-ical work is needed on these organisms. Some of the mostinteresting and unanticipated advances are occurring in re-search on organisms that have multiple copies of chemotaxisgenes and those that have employed, for controlling develop-ment, proteins that once had a chemotaxis function. These newareas, as well as the dynamics of receptor-receptor interactionsin bringing about extreme sensitivity to the slightest changes inattractant concentrations over many orders of magnitude, arepromising areas of future investigation.

Understanding the structural changes that underlie this re-markable capability is a major challenge; however, we believethat the talented cadre of investigators are up to meeting thischallenge. All of these new investigations should serve to makethe study of bacterial chemotaxis as exciting during the next 30years as it has been during the past 30.

ACKNOWLEDGMENT

This work was supported by National Institutes of Health grant RO1GM54365.

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